PROJECT SUMMARY Expansion of short nucleotide repeats causes a variety of neurological disorders. While the pathological mechanism of each of these disorders has been studied individually, converging evidence suggests that two unusual molecular hallmarks are shared among many RNA repeats transcribed from the expanded repeat genomic loci. These unusual molecular hallmarks are (1) the formation of large ribonucleoprotein (RNP) structures known as RNA foci and (2) a noncanonical type of translation known as repeat-associated non- AUG-initiated translation or RAN translation. Currently, the molecular features of RNA repeats that promote these unusual properties, the cellular factors that mediate these properties, and the relative contributions of each of these two properties to cellular toxicity remain unclear. This proposal aims to develop a suite of high- throughput methods to synthesize and determine the biochemical and cellular properties of a large repertoire of RNA repeat sequence variants, with the goal of identifying the sequence/structural features of RNA repeats that promote RAN translation and/or RNA foci formation. Regulatory sequence elements within the upstream flanking region of the repeats will also be determined. Cellular proteins that specifically interact with repeat variants that are RAN-translated or those that form RNA foci will be identified using RNP complex purification followed by quantitative proteomics, and their potential roles in RAN translation and/or RNA foci formation will be assessed. The identification of molecular features and cellular factors that promote these unusual RNA repeat-associated properties will potentially enable (1) the disassociation of the cellular toxicity contributed by RNA foci and RAN translation and (2) the identification of the physiological counterpart of RAN translation, namely, non-pathogenic endogenous RNAs that undergo non-AUG-initiated translation under physiological conditions. These analyses will deepen our understanding of the molecular basis of the unique properties associated with pathogenic RNA repeats, and potentially inspire new strategies for therapeutic intervention of repeat expansion disorders.